Transcription factors are important regulators of gene expression. Gene-specific transcription factors provide a promising class of targets for novel therapeutics directed to human diseases. For example, transcription factors offer substantial diversity. Over 300 gene-specific transcription factors have been described, and the human genome may encode as many as 3000. Transcription factors also offer substantial specificity. Each and every factor offers unique molecular surfaces to target. Additionally, transcription factors are known to be involved in a wide variety of human diseases. For example, many tumors are associated with the activation of a specific oncogene. A third of known proto-oncogenes and three fourths of all anti-oncogenes are transcription factors. Transcription factors involved in cancers, such as leukemias and lymphomas, include Ets and Stat related transcription factors.
Signal transduction pathways often effect changes in cellular biology by inducing programs of gene expression through the activation of transcription factors. NF-KB is a transcription factor in that it is activated by many diverse stimuli that alert a cell or organism to stressful or infectious conditions and activate a response (Baldwin, A. S., Jr., Annu Rev Immunol 14:649–83 (1996); Ghosh, S., et al., Annu Rev Immunol 16:225–60 (1998)). These include UV and γ-irradiation, bacterial and viral products (e.g., lipopolysaccaride and dsRNA), proinflammatory cytokines (e.g., TNFα and IL-1), antigen recognition by the T cell and B cell receptor complexes, and apoptotic and necrotic stimuli. NF-KB regulates a multitude of genes involved in the development and function of the immune response, inflammation, cell growth control, and in antiapoptotic responses (Pahl, H. L., Oncogene 18:6853–66 (1999)). In addition, several viruses use NF-KB to regulate viral gene expression, including HIV, HSV, EBV, CMV and Adenovirus. Moreover, activiation of this transcription factor contributes to the resistance of some tumors to chemotherapeutic agents.
Many stimuli activate NF-KB by causing the phosphorylation and destruction of IKBS, inhibitory molecules that bind NF-KB in the cytoplasm. The signal-induced phosphorylation of IKB occurs at two specific serines in its N-terminus and is accomplished by the IKK complex, which is composed of two kinase subunits, IKKα and IKKβ, and a noncatalytic subunit, NEMO/IKKy (Karin, M. & Y. Ben-Neriah, Annu Rev Immunol 18:621–63 (2000)). Subsequent to phosphorylation, IKB is ubiquitinated and degraded by the 26S proteasome, leaving NF-KB free to translocate to the nucleus to activate target genes.
Several pathways that activate NF-KB employ a set of signaling molecules that link stimulus recognition to IKK complex activation. These include cell surface receptors which recognize ligands, proximal kinases that may directly phosphorylate and activate the IKK complex, and adapter proteins which physically link ligand-bound receptor complexes to kinase activation. The IKK complex is activated by many stimuli, but the mechanism has not been reported on.
Current methods for the identification of molecules that modulate activity of a transcription factor include yeast two-hybrid screening using a particular bait molecule. The bait molecule is usually a molecule known to modulate activity of the transcription factor. Another method is the biochemical purification of proteins physically associated with a particular molecule known to modulate activity of a transcription factor. These two methods have inefficiencies because they use as the detection method a binding property, i.e. the ability to bind to a particular target molecule which itself is a component of the signaling pathway for activating the transcription factor. Thus, these methods detect proteins which can bind to a particular target molecule, whether or not the detected protein actually participates in regulation of the transcription factor. These methods are also limited by what bait/target molecule is used for detecting associated proteins.
Another current approach to identifying transcription factor modulators is the biochemical purification of proteins which possess specific catalytic properties. These specific catalytic properties have been determined to be critical steps in one or more pathways that modulate activity of a particular transcription factor. This method is limited in that it only detects molecules with specific catalytic functions. For example, if one uses this method to detect and isolate a kinase for IKB (the inhibitor of the transcription factor NF-KB), one would only find kinase molecules by this method.
Yet another current approach is the cloning of genes homologous to molecules known to modulate activity of a transcription factor. This method is limited in that it only detects genes/proteins with particular homology. For example, if one uses this method to detect (by conventional low stringency screening of cDNA libraries) homologs of TRAF2, a known activator of NF-KB, one would only find members of the TRAF family of proteins in the screen.
Therefore, it is desirable to provide new and more efficient methods to identify molecules which participate in signaling pathways involving transcription factors. It is further desirable to identify and provide therapeutic targets for disease states involving altered activity of transcription factors.